In response to the possible release of smallpox (variola) by bioterrorists, vaccinia virus (VV) vaccination of an at-risk population is the only available prophylactic intervention. However, the vaccine has known complications, especially in immunocompromised hosts, pregnant women and infants. In past vaccination efforts, such complications were treated in the U.S. with vaccinia human immune globulin (VIG) obtained from VV immunized people. Although this material had efficacy, little is known about which components of the immune globulin were effective and there could be batch-to-batch variation in efficacy. Moreover, since routine vaccination has been discontinued for many years, there is only a limited supply of VIG available, and there are concerns about its safety. Our long-term goal is to develop a cocktail of defined and high affinity antibodies to VV proteins that will replace the use of traditional VIG in the event that mass VV vaccination is needed. Our strategy is to develop monoclonal antibodies to various VV envelope proteins that are known to provoke a neutralizing and/or protective response in a mouse model. A cocktail of such antibodies, which we will call VIG-R (replacement) will provide a uniform and secure source of a VV immune therapeutic reagent. In this application, we propose experiments in four specific aims. These are: 1) to express and characterize VV envelope proteins LIR, A33R and B5R in a baculovirus expression system and to prepare mouse and rabbit antibodies to those proteins; 2) to develop and characterize human monoclonal antibodies to LIR, A33R and BSR produced using phage display; and 3) to test the ability of immune reagents against VV proteins to protect mice from challenge with VV. Fulfillment of the aims of this grant should provide new information about each of the VV proteins and antibodies, as well as a source of reagents that can comprise a VIG-R. If our approach appears promising, we will expand this study to include other VV proteins. The reagents developed in this grant application will also be valuable for basic studies concerning the role of these proteins in the VV life cycle and could themselves be considered as vaccine candidates in future investigations.